Method and apparatus for the detection and classification of microorganisms in water

ABSTRACT

The method and apparatus for detecting the presence of an organism in a sample of liquid involves collecting an extinction spectrum of liquid sample, deconvoluting the spectrum to obtain a particle size distribution for the sample, comparing the spectrum and the particle size distribution with, respectively, a control spectrum and a control particle size distribution for the organism, and determining from the comparisons whether the organism to be detected is present in the sample. The apparatus and method permit on-line real-time quantitative classification, identification, and viability assessment of an organism such as a harmful microorganism in a water supply line.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates no the detection of organisms in liquids,and, more particularly, to the spectrophotometric detection ofmicroorganisms in aqueous solutions.

2. Description of Related Art

Water quality is an extremely important environmental issue,particularly as regards the quality of drinking water. The number andsize of the particulate matter suspended in drinking water iscontinuously monitored after the filtration units in water treatmentfacilities, specifically to detect the presence of microorganisms suchas enteroviruses, protozoa, and bacteria capable of presenting serioushealth hazards.

The enteric protozoa Cryptosporidium and Giardia are known to causewaterborne diseases, even when present in fairly dilute concentrations.Giardia is the most frequently identifiable agent responsible for suchdiseases in the United States; Cryptosporidium has caused outbreaks inthe United Kingdom as well as the United States and is now recognized asbeing one of the most disinfectant-resistant waterborne pathogens known.These occurrences have emphasized the need for rapid detectiontechniques or contaminants in source and treated water.

Currently there is no on-line instrumentation capable of detecting,counting, and classifying specific microorganisms. The technology knownin the art requires that the particulate matter suspended in the waterbe concentrated and then detected with the use of microscopictechniques. Such laboratory techniques include immunofluorescent assay(IFA), polymerase chain reaction (PCR), flow cytometry (FC), and cellsorting. IFA technology may be used in conjunction with microscopy foridentification following labeling with specific antibodies. Thelimitations associated with IFA include long analysis times, aninability to detect viability and to distinguish between species, andlow sensitivity. The disadvantages of PCR include environmentalinterference, long analysis times, and an inability to quantifyorganisms. FC has the disadvantages of high instrumentation costs, highlevel of training of personnel required, an inability to distinguishbetween species, and small sample volumes.

In industrial settings, detection methods include turbidity and particlecounting. Turbidity is known as a technique for evaluating filterefficiency and water quality and can be used on line. Standard turbiditymeasurements respond to both particle size and number; therefore, theydo not distinguish between the two. Liquid-borne particle counters (LPC)illuminate a very small sample volume for analysis and have nottraditionally been used for on-line applications. Although LPC cannotdifferentiate between species, if coupled with adequate samplingstrategies, particle counters can be used effectively for on-lineapplications.

As is known from spectroscopy theory, a measure of the absorption of asolution is the extinction coefficient, which also provides a measure ofthe turbidity. Spectra in the visible region of the electromagneticspectrum reflect the presence of metal ions and large conjugatedaromatic structures double-bond systems. In the near-uv region smallconjugated ring systems affect absorption properties. However,suspensions of very large particles are powerful scatterers ofradiation, and in the case of microorganisms, both light scattering andabsorption effects are sufficiently strong to permit quantitativedetection and classification. It is therefore known to use uv/visspectroscopy to monitor purity, concentration, and reaction rates ofsuch large molecules.

Many attempts have been made to estimate the PSD and the chemicalcomposition of suspended particles using optical spectral extinction(turbidity) measurements. However, previously used techniques requirethat either the form of the PSD be known a priori or that the shape ofthe PSD be assumed. The present inventor has applied standardregularization techniques to the solution of the turbidity equation andhas demonstrated correct PSDs of a large variety of polymer lattices,protein aggregates, silicon dioxide particles, and microorganisms.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide aspectroscopic and turbidimetric technique for the identification andclassification of microorganisms in a liquid medium.

It is a further object to provide on-line instrumentation capable ofdetecting, counting, and classifying particulates in an aqueous medium.

It is an additional object to provide such instrumentation having atleast 2 nanometer resolution.

It is another object to provide a technique capable of detecting theviability of microorganisms present in a liquid medium.

It is yet a further object to provide a monitoring technique fordrinking water to prevent the passage of waterborne pathogens into thedrinking water supply.

These and other objects are addressed by the apparatus and method of thepresent invention for detecting the presence of an organism in a sampleof liquid. The method comprises the steps of collecting an extinctionspectrum over a predetermined range of wavelengths of the sample ofliquid and then deconvoluting the spectrum to obtain a particle sizedistribution for the sample. The wavelength range generally comprisesthe entire ultraviolet/visible (uv/vis) range, from 180 to 900 nm. In aparticular embodiment, however, the spectrum is collected over thewavelength range of 400 to 820 nm. Previously used systems have used asingle frequency to classify organisms, whereas in the present inventionthe whole spectrum is solved to obtain a self-consistent solution. Thespectrum and the particle size distribution are then compared with,respectively, a control spectrum and a control particle sizedistribution for the organism. From these comparisons it can bedetermined from the comparisons whether the organism to be detected ispresent in the sample.

An additional embodiment of the method, used for particle countingapplications, further comprises the step of determining the quantity ofthe organism to be detected in the sample from the particle sizedistribution.

Yet another embodiment entails determining the viability of the organismto be detected in the sample from the particle size distribution.

The apparatus of the present invention comprises means for performingthe above-listed steps. In a particular embodiment, the spectrumcollecting means comprises a spectrophotometer.

The features that characterize the invention, both as to organizationand method of operation, together with further objects and advantagesthereof, will be better understood from the following description usedin conjunction with the accompanying drawing. It is to be expresslyunderstood that the drawing is for the purpose of illustration anddescription and is not intended as a definition of the limits of theinvention. These and other objects attained, and advantages offered, bythe present invention will become more fully apparent as the descriptionthat now follows is read in conjunction with the accompanying drawing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows extinction spectra for Cryptosporidium parvum and Giardialamblia.

FIG. 2 shows extinction spectra for Cryptosporidium parvum indirectlystained with ab-FITC.

FIG. 3 shows extinction spectra for Giardia lamblia indirectly stainedwith ab-FITC.

FIG. 4 shows extinction spectra for Cryptosporidium parvum directlystained with ab-rhodamine.

FIG. 5 shows extinction spectra for Giardia lamblia directly stainedwith ab-rhodamine.

FIG. 6 is a weight-based particle size distribution of C. parvum and C.baileyi.

FIG. 7 is a weight-based particle size distribution of G. lamblia and G.muris.

FIG. 8 is a block diagram of the system configuration.

FIG. 9 is a flow chart of the data analysis method.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A description of the preferred embodiments of the present invention willnow be presented with reference to FIGS. 1-9.

The apparatus 10 and method of using same for detecting the presence ofan organism in a sample of liquid are depicted in FIG. 8. In theparticular embodiment to be treated herein for microorganisms, theprotozoa Cryptosporidium parvum and Giardia lamblia in aqueousdispersion are detected through spectroscopic means over a predeterminedwavelength range. In this embodiment, shown in FIG. 8 for the detectionof the protozoa at a water treatment plant 30 having a filtration unit40, a uv/vis extinction spectrum is collected with a pair ofspectrophotometers/turbidimeters 20, one upstream of the filtration unit40 and one downstream of the filtration unit 40 over the predeterminedwavelength range is generally 400-820 nm. Three exemplaryspectrophotometers that may be utilized comprise an Ocean Optics S-1000miniature diode array spectrophotometer, a Hewlett-Packard 8452A diodearray spectrophotometer, and a Perkin Elmer 3840 uv/vis spectrometer.

The measurements reported herein were conducted at room temperature in a1-cm-path-length cell. Prior to collecting a spectrum, it should bedetermined whether the water sample is in a linear range of thespectrophotometer 20. If necessary, the water sample is diluted with abackground solution to reach the linear range of the spectrophotometer20. The data are then transmitted to a computer 50, which is configuredin this exemplary embodiment to perform analytical tasks such as dataprocessing, to issue alarms, and to issue signals for control action.

A spectrum obtained in this manner is then corrected for background. Theaverage refractive index of the protozoa was approximated as 1.386 andwas assumed constant as a function of wavelength. The refractive indexof water as a function of wavelength was calculated from previouslypublished data.

Exemplary extinction spectra of Giardia lamblia and Cryptosporidiumparvum are shown in FIG. 1. C. parvum is known to be spherical and tohave a size of approximately 4-5 μm; G. lamblia is ovoid, with a lengthof approximately 10-15 μm and a width of 6-10 μm. These spectra aretypical of purified samples diluted with formalin.

A tail trailing toward longer wavelengths can be seen in FIG. 1,indicating the presence of particulate matter. The greater intensity ofthe G. lamblia spectrum as compared with that of C. parvum is due to thegreater scattering caused by the greater size of G. lamblia.

Comparison of the spectra in the wavelength region 200-400 nm indicatesthat there are spectral features that are usable for identification andclassification purposes. It is known that staining a microorganism priorto collecting the spectrum can improve the sensitivity of spectroscopymeasurements. Two stains that have been utilized on the protozoa underexamination are ab-FITC complex and ab-rhodamine.

FIGS. 2 and 3 show the extinction spectra of C. parvum and G. lamblia,respectively, indirectly stained with ab-FITC complex, which fluorescesapple-green (the wavelength is in the general range of 300 nm; however,scattering and absorption will cause a shift in the pattern, makingthese numbers approximate). FITC has been known in the art for use bymicroscopists and can enable detection of fewer than ten organisms permilliliter from a purified stock. A comparison of the spectra in FIG. 1with those in FIGS. 2 and 3 clearly indicates that staining with ab-FITCenhances the sensitivity of the spectroscopic measurements. In addition,the use of staining allows a direct comparison between the presentresults and previous microscopy analysis. It can also be noted thatother features of the spectra are retained, with G. lamblia having ahigher intensity in the visible range, due to its larger size.

FIGS. 4 and 5 show the extinction spectra of C. parvum and G. lambliastained with ab-rhodamine. This stain, which is red (as above forab-FITC, an approximate wavelength range is generally 400 nm), is knownfor use in microscopy studies. A comparison of FIGS. 1-5 indicates thatthe distribution of stains is different for the two protozoa and that,in addition to enhancing the signal, the use of stains greatlyfacilitates the identification and classification of microorganisms.

The collection of spectra such as shown in FIGS. 1-5 therefore permitsan identification of microorganisms based on spectral signature; thatis, the sample spectrum can be compared with a control spectrum for theorganism to determine whether the organism is present in the watersample.

A spectrum collected as discussed above is then analyzed with the use ofcomputation means 50, which comprises a computer having software fordeconvoluting the spectrum to obtain a particle size distribution forthe sample.

The present inventor has devised a technique for determining adiscretized particle size distribution from turbidity spectra, showndiagrammatically in FIG. 9. The equations providing the theoreticalframework are developed from a relation between the turbidity as afunction of wavelength τ(λ₀) and the normalized particle sizedistribution f(D): ##EQU1## where D is the effective particle diameter,Q(λ₀, D) corresponds to the Mie scattering coefficient, and N_(p) is thenumber of particles per unit volume. Equation (1) can be written inmatrix form by discretizing the integral with an appropriate quadratureapproximation:

    τ=A f+ε,                                       (2)

where ε represents both experimental errors and errors due to the modeland the discretization procedure. The regularized solution to Eq. (2) isgiven by:

    f(γ)=(A.sup.T A+γH).sup.-1 A.sup.T τ,      (3)

where H is a covariance matrix that essentially adaptively filters theexperimental and the approximation errors (ε), and γ is theregularization parameter estimated using the generalizedcross-validation technique. This technique requires the minimization ofthe following objective function with respect to γ:

    V(γ)=m|[I-A(A.sup.T A+γH).sup.-1 ]τ|.sup.2 /Tr{[I-A(A.sup.T A+γH).sup.-1 ]A.sup.T }.sup.2(4)

A simultaneous applications of Eqs. (3) and (4) to the measuredturbidity spectra yields the discretized particle size distribution. Thecorrected scattering spectra can then be used for composition analysisand/or to fingerprint the absorption characteristics of particles.

Quantitative information, such as shown in FIGS. 6 and 7, may beobtained using the method of the present invention from the use of Eqs.(3) and (4) and spectra such as shown in FIG. 1.

FIG. 6 shows a weight-based particle size distribution (PSD) for C.parvum (peak 2) and C. baileyi (peaks 1 and 3). Peaks 2 and 3 arecentered around 4.8 μm, the size normally associated with C. parvum. Thedifference between the two samples is seen at peak 1, which is centeredat 2.5 μm. By deconvoluting the extinction spectra of the purifiedsamples, the PSD was obtained. The abscissa represents the diameter ofthe microorganisms, and the ordinate, the weight-based density. Peak 1is indicative of cell debris from broken oocysts and sporozoites; the C.parvum sample was purified by high-performance liquid chromatography,whereas the C. baileyi sample was not.

FIG. 7 shows the PSD for G. muris (peaks 1, 4, and 6), and G. lamblia(peaks 2, 3, and 5). Peaks 1 and 2, approximately located at 2.8 μm, areindicative of cell debris resulting from the preparation of the samples.Peaks 3 and 4 are located at approximately 9.0 μm and correspond totypical dimensions of both G. lamblia and G. muris. Peaks 5 and 6,centered at approximately 16 μm, correspond to cell aggregates and/orcells oriented perpendicular to the incident light.

Utilizing the above method to calculate the particle size distribution,the PSD can then be compared with a control particle size distributionfor the organism, and then one can determine from the comparison whetherthe organism to be detected is present in the sample.

These calculations can also be used to determine the quantity of theorganism to be detected in the sample from the particle sizedistribution.

Another embodiment of the method of the present invention comprisesdetermining the viability of the organism to be detected in the samplefrom the particle size distribution. In this embodiment staining wouldagain be used, with the stain uptake providing an indication of particleviability, for instance. This technique could also be performed on-line.

A particular use for the apparatus and method of the present inventionis for on-line, real-time measurements to detect the presence of anorganism in a flowing liquid. In order to accomplish this, a sample istaken from the flowing liquid, and an extinction spectrum is collectedover a predetermined range of wavelengths of the sample of liquid, thespectrum being collected at the site of the sample collection. Stainingmay also be used as described above.

As previously the spectrum is deconvoluted to obtain a particle sizedistribution for the sample, and the spectrum and the particle sizedistribution are compared with, respectively, a control spectrum and acontrol particle size distribution for the organism. From thesecomparisons it can be determined whether the organism to be detected ispresent in the sample.

Also as previously, the quantity and the viability of the organism to bedetected in the sample can be determined from the particle sizedistribution.

It is contemplated that such an on-site apparatus could be placed incommunication with virtually any water line, including at an entrance toa dwelling. Should harmful particles be detected, an alarm 60 couldring, as shown in FIG. 8 for a water treatment plant 30, indicating thatthe water flowing through the system was unsafe. Corrective action 70would then be taken, such as shutting down the water flow at the plant,and the water could be subjected to additional purification prior toproceeding downstream.

It may be appreciated by one skilled in the art that additionalembodiments may be contemplated, including systems and methods fordetecting other types of organisms and particles in a liquid, forstudying and monitoring organism life cycles, for examining drugdelivery mechanisms, for studying the mechanisms of infectious diseases,and for detecting abnormal cells.

In the foregoing description, certain terms have been used for brevity,clarity, and understanding, but no unnecessary limitations are to beimplied therefrom beyond the requirements of the prior art, because suchwords are used for description purposes herein and are intended to bebroadly construed. Moreover, the embodiments of the apparatusillustrated and described herein are by way of example, and the scope ofthe invention is not limited to the exact details of construction.

Having now described the invention, the construction, the operation anduse of preferred embodiment thereof, and the advantageous new and usefulresults obtained thereby, the new and useful constructions, andreasonable mechanical equivalents thereof obvious to those skilled inthe art, are set forth in the appended claims.

What is claimed is:
 1. A method for detecting the presence of anmicroorganism in a sample of liquid, the method comprising the stepsof:collecting an extinction spectrum over a predetermined range ofwavelengths of the sample of liquid; deconvoluting the spectrum toobtain a particle size distribution for the sample; comparing thespectrum and the particle size distribution with, respectively, acontrol spectrum and a control particle size distribution for themicroorganism; and determining from the comparisons whether themicroorganism to be detected is present in the sample.
 2. The method fordetecting the presence of an microorganism recited in claim 1, furthercomprising the step of determining the quantity of the microorganism tobe detected in the sample from the particle size distribution.
 3. Themethod for detecting the presence of an microorganism recited in claim1, further comprising the step of determining the viability of themicroorganism to be detected in the sample from the particle sizedistribution.
 4. The method for detecting the presence of anmicroorganism recited in claim 1, wherein the collecting step comprisescollecting an extinction spectrum over a wavelength range of 400 to 820nm.
 5. A method for spectrophotometrically detecting the presence of amicroorganism in a water sample, the method comprising the stepsof:determining whether the water sample is in a linear range of thespectrophotometer; diluting the water sample if necessary with abackground solution to reach the linear range of the spectrophotometer;using the spectrophotometer to collect a turbidity spectrum of the watersample over the wavelength range of 400 to 820 nm; correcting thespectrum for background; calculating a particle size distribution fromthe corrected spectrum; comparing the particle size distribution with aknown particle size distribution for the microorganism to be detected;and determining from the comparison whether the microorganism is presentin the water sample.
 6. The method recited in claim 5, furthercomprising the step of staining the microorganism prior to collectingthe turbidity spectrum for improving sensitivity.
 7. The method recitedin claim 5, further comprising the step of quantifying, from theparticle size distribution, the number of microorganisms present in thewater sample.
 8. The method recited in claim 5, further comprising thestep of determining, from the particle size distribution, the viabilityof the microorganisms present in the water sample.